The fact that individual cardiac cells can express hundreds of microRNAs, each with potentially hundreds of targets, raises the question of how such a complex mode of regulation can possibly achieve specificity. We hypothesize that this occurs through the ability of individual 3'UTRs to interact with multiple microRNAs simultaneously, such that concurrent binding of two (or more) microRNAs is required for biologically meaningful regulation. Using a method that we developed to identify microRNA-mRNA interactions biochemically, we showed that miR-1 and miR-133a associate simultaneously with the 3'UTR of the cardiac transcription factor, Hand2. This is probably the best example to date in any system of concurrent binding by two microRNAs to a single target. We hypothesize that binding of miR-1 and miR-133a to the Hand2 3'UTR is mutually interdependent, such that both microRNAs must associate with their recognition elements (MREs) to achieve efficient repression. Such a mechanism would increase signaling complexity, generating specificity of targeting and constraining the number of effective mRNA targets for an individual microRNA. This scenario may be characteristic of other microRNA targets, and we will use a new assay developed in our lab to identify additional cardiac mRNAs that are subject to a similar dual mode of regulation. These studies could answer a seminal question in microRNA signaling-how the multitude of microRNAs and predicted targets can achieve specificity. To accomplish our goals, we will determine how miR-1 and miR-133a cooperate to regulate Hand2 expression. Our data indicates that binding of miR-1 and miR-133a is mutually interdependent, which has never before been described. We will elucidate the functional implications of this finding by utilizing a set of novel bidirectional ratiometric sensors. To elucidate the mechanism underlying the interdependency of miR-1 and miR-133a binding, we will target Ago2, a core component of the RNA induced silencing complex (RISC), to the mutated Hand2 miR-1 or miR-133a MREs to determine whether recruitment of Ago2 and associated proteins can rescue effects of the MRE mutations. We will also determine whether the coordinate regulation by miR-1 and miR-133a is shared by other cardiac mRNAs. Bioinformatic algorithms designed to predict microRNA targets are notoriously imprecise in their ability to identify authentic interactions, missing many interactions and falsely predicting others. We developed a novel approach for identifying these targets that employs a dominant negative RISC component to trap microRNA-mRNA intermediates prior to degradation. We will use this approach to identify other cardiac mRNAs that associate with both miR-1 and miR-133a and test whether microRNA binding and function are similarly interdependent. Understanding how combinations of microRNAs affect expression of targets is essential for developing effective microRNA-based therapies. PUBLIC HEALTH RELEVANCE: This project uses several novel approaches to examine the role of micro RNA pathways in regulating cardiac transcription factors with implications for human congenital and acquired heart disease. Characterization of these pathways may identify new therapies for treating cardiovascular disease.